Phenolic Compounds and their Properties
Phenolic compounds (also called phenolics), or polyphenols, constitute one of the most numerous and widely-distributed groups of substances in the plant kingdom, with more than 8,000 phenolic structures currently known. Polyphenols are products of the secondary metabolism of plants. The expression “phenolic compounds” embraces a considerable range of substances that possess an aromatic ring bearing one or more hydroxyl substituents. Most of the major classes of plant polyphenol are listed in Table 1, according to the number of carbon atoms of the basic skeleton. The structure of natural polyphenols varies from simple molecules, such as phenolic acids, to highly polymerized compounds, such as condensed tannins (HARBORNE J B (1980) Plant phenolics. In: BELL E A, CHARLWOOD BV (eds) Encyclopaedia of Plant Physiology, volume 8 Secondary Plant Products, Springer-Verlag, Berlin Heidelberg New York. Pp: 329-395).
The three important groups for humans are phenolic acids (C6-C1, C6-C2 and C6-C3), flavonoids (C6-C3-C6) and high-molecular-weight polyphenols (more than 30 carbon atoms). Indeed, the phenolics, particularly polyphenols, exhibit a wide variety of beneficial biological activities in mammals, including antiviral, antibacterial, immune-stimulating, antiallergic, antihypertensive, anti-ischemic, antiarrhythmic, antithrombotic, hypocholesterolemic, antilipoperoxidant, hepatoprotective, anti-inflammatory, anticarcinogenic, antimutagenic, antineoplastic, anti-thrombotic and vasodilatory actions. They are powerful antioxidants in vitro.
TABLE IThe major classes of phenolic compounds (or phenolics) in plants(HARBORNE JB, 1980)NUMBEROFCARBONBASICATOMSSKELETONCLASSEXAMPLES6C6Simple phenolsCatechol, hydroquinoneBenzoquinones2,6-Dimethoxybenzoquinone7C6-C1Phenolic acidsGallic, salicylic8C6-C2Acetophenones3-Acetyl-6-Tyrosine derivativesmethoxybenzaldehydePhenylacetic acidsTyrosolp-Hydroxyphenylacetic9C6-C3HydroxycinnamicCaffeic, ferulicacidsMyristicin, eugenolPhenylpropenesUmbelliferone, aesculetinCoumarinsBergenonIsocoumarinsEugeninChromones10C6-C4NaphthoquinonesJuglone, plumbagin13C6-C1-C6XanthonesMangiferin14C6-C2-C6StilbenesResveratrolAnthraquinonesEmodin15C6-C3-C6FlavonoidsQuercetin, cyanidinIsoflavonoidsGenistein18(C6-C3)2LignansPinoresinolNeolignansEusiderin30(C6-C3-C6)2BiflavonoidsAmentoflavonen(C6-C3)nLignins(C6)nCatechol melanins(C6-C3-C6)nFlavolans(CondensedTannins)
Among the phenolic acids, the most important constitutive carbon frameworks are the hydroxybenzoic (C6-C1) and hydroxycinnamic (C6-C3) structures. The hydroxybenzoic acid content of edible plants is generally very low, with the exception of certain red fruits, black radish and onions, which can have concentrations of several tens of milligrams per kilogram fresh weight. Hydroxybenzoic acids are components of complex structures such as hydrolyzable tannins (gallotannins in mangoes and ellagitannins in red fruits such as strawberries, raspberries and blackberries). The hydroxycinnamic acids are more common than are the hydroxybenzoic acids and consist chiefly of p-coumaric, caffeic, ferulic and sinapic acids. These acids are rarely found in the free form, except in processed food that has undergone freezing, sterilization or fermentation. The bound forms are glycosylated derivatives or esters of quinic acid, shikimic acid and tartaric acid. Caffeic acid and quinic acid combine to form chlorogenic acid, which is found in many types of fruit and in high concentration in coffee. Caffeic acid, both free and esterified, is generally the most abundant phenolic acid and represents between 75% and 100% of the total hydroxycinnamic acid of most fruit (MANACH C, SCALBERT A, MORAND C, REMESY C, JIMENEZ L (2004) Polyphenols: food sources and bioavailability. Am J Clin Nutr 79: 727-747).
The flavonoids consist of a large group of low-molecular weight polyphenolic substances, benzo-γ-pyrone derivatives, that are diverse in chemical structure; they represent the most common and widely distributed group of plant phenolics. The flavonoids' common structure is that of diphenylpropanes (C6-C3-C6); it consists of two aromatic rings (cycles A and B) linked through three carbons that usually form an oxygenated heterocycle (cycle C). FIG. 1 shows the basic structure and the system used for the carbon numbering of the flavonoid nucleus. Structural variations within the rings subdivide the flavonoids into several families: flavonols, flavones, flavanols, isoflavones, antocyanidins and others. These flavonoids often occur as glycosides, glycosylation rendering the molecule more water-soluble and less reactive toward free radicals. The sugar most commonly involved in glycoside formation is glucose, although galactose, rhamnose, xylose and arabinose also occur, as well as disaccharides such as rutinose. The flavonoid variants are all related by a common biosynthetic pathway, incorporating precursors from both the shikimate and the acetate-malonate pathways (CROZIER A, BURNS J, AZIZ A A, STEWART A J, RABIASZ H S, JENKINS G I, EDWARDS C A, LEAN MEJ (2000) Antioxidant flavonols from fruits, vegetables and beverages: measurements and bioavailability. Biol Res 33: 79-88). Further modifications occur at various stages, resulting in an alteration in the extent of hydroxylation, methylation, isoprenylation, dimerization and glycosylation (producing O- or C-glycosides). Phenolic compounds act as antioxidants with mechanisms involving both free radical scavenging and metal chelation. Indeed, excess levels of metal cations of iron, zinc and copper in the human body can promote the generation of free radicals and contribute to the oxidative damage of cell membranes and cellular DNA; by forming complexes with these reactive metal ions, they can reduce their absorption and reactivity. It has to be underlined that although most flavonoids chelate Fe2+, there are large differences in the chelating activity. In particular, the dihydroflavonol taxifolin chelates more efficiently Fe2+ than the corresponding flavonol quercetine (VAN ACKER SABE, VAN DEN BERG D J, TROMP MNJL, GRIFFIOEN DHG, VAN BENNEKOM, VAN DER VIJGH WJF, BAST A (1996) Structural aspects of antioxidant activity of flavonoids. Free Radic Biol Med 20: 331-342).
Flavonoids have ideal structural chemistry for free radical-scavenging activities (several studies have shown the flavonoids to act as scavengers of superoxide anions, singlet oxygen, hydroxyl radicals and lipid peroxyl radicals by rapid donation of a hydrogen atom). One important finding from the studies of the relationship between the structural characteristics of flavonoids and their antiradical activity is that a catechol moiety (3′, 4′-dihydroxyphenol) on ring B is required for good scavenging activity. Recently, this statement was confirmed with, nevertheless, a modulation: in a study about the relationship between the structural characteristics of 29 flavonoids and their antiradical activity, it was indeed observed that the catechol structure in the B ring is not always a conditio sine qua non in achieving high free radical-scavenging activity and that highly active flavonoids possess a 3′,4′-dihydroxy B ring and/or a 3-OH group (AMIC D, DAVIDOVIC-AMIC D, BESLO D, TRINAJSTIC N (2003) Structure-radical scavenging activity relationships of flavonoids. Croatica Chem Acta 76: 55-61). Flavonoids have been shown to be more effective antioxidants in vitro than vitamins E and C on a molar basis (RICE-EVANS C A, MILLER N J, PAGANGA G (1997) Antioxidant properties of phenolic compounds. Trends in Plant Science 2: 152-159). There are also reports of flavonoids inhibiting the activity of enzymes such as oxygenases.
It must be underlined that the hydrophobicity of polyphenols is intermediate between that of vitamin C (highly hydrophilic) and that of vitamin E (highly hydrophobic); polyphenols are thus expected to act at water-lipid interfaces and may be involved in oxidation regeneration pathways with vitamins C and E.
Phenolic Derivatives and their Preparation
Due to their low aqueous solubility and/or high sensitivity toward oxidation, the use of phenolics in pharmaceutical or cosmetic preparations requires adapted and specific formulations. Since these formulations must also satisfy the constraints associated with their final usage, the compromise between acceptability, concentration and stability is often difficult to reach.
More water-soluble and/or oxidation-resistant forms of phenolics such as the glycosides are not always available in nature and may demand, when they exist, complex procedures of extraction and purification from the plant material. Both chemical and biochemical (enzymatic) approaches have been attempted to increase water solubility and/or stability. As phenolic compounds have several free hydroxyl groups, attempts for chemical modifications of phenolic compounds lead to unselective reactions, generating a panel of different molecules. Further steps of purification are then required to recover the desired product(s).
As far as the biochemical approach is concerned, three ways have been investigated to date to obtain phenolic glycosides and basically flavonoid glycosides.
The first way relies on glycosyltransferases able to transfer the sugar moiety of a sugar nucleotide to an acceptor (in the case of UDP-glucose:glucosyltransferases (UGT), glucose is transferred from uridine 5′-diphosphoglucose). These enzymes, which contribute to the synthesis of secondary metabolism in plants, have broad acceptor substrate specificities (LIM E K, HIGGINS G S, BOWLES D J (2003) Regioselectivity of glucosylation of caffeic acid by UDP-glucose:glucosyltransferase is maintained in planta. Biochem J 373: 987-92; LIM E K, ASHFORD D A, HOU B, JACKSON R G, BOWLES D J (2004) Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides. Biotechnol. Bioeng. 87(5): 623-31). Nevertheless, this approach is impaired by the very high cost of the sugar nucleotides and the regeneration of the sugar nucleotide substrate, which is a way to decrease the substrate cost, is difficult to master on a large scale.
The second way relies on saccharide-transferring enzymes able to transfer glucose from an α-glucosyl saccharide. Said enzymes are selected from the hydrolases α-glucosidase (EC 3.2.1.20) and α-amylase (EC 3.2.1.1), and from the transferase cyclodextrin-glucanotransferase (EC 2.4.1.19). Their substrates are amylose, dextrins, cyclodextrins, maltooligosaccharides and partial starch hydrolysates, all of them containing mainly or exclusively glucosyl residues linked to each other through an α 1→4 osidic bond. According to this approach, U.S. Pat. No. 5,565,435 states that α-glucosyl quercetin is obtained. It has to be underlined that the starch-degrading enzymes link the glucosyl residue to the flavonoid through an α-osidic bond whereas the UDP-glucose:glucosyltransferase investigated by LIM et al. links the glucosyl residue to the flavonoid through a β-osidic bond. It has also to be underlined that in the conditions described in U.S. Pat. No. 5,565,435, the quercetin molecule could be solubilized by adjusting the pH to 8.5 and by maintaining the reaction medium at 60° C. The solubilisation of phenolics in alkaline media is due to the formation of phenolates; in these pH and temperature conditions, the stability of the substrate was achieved by operating under anaerobic conditions. It thus appears that this mode of preparation is highly difficult to control and manage and that a simple mode of preparation should be valuable.
The third way involves glucosyltransferases using sucrose β-D-fructofuranosyl-α-D-glucopyranoside) as a glucosyl donor and producing glucans with the release of fructose. Several attempts have been achieved with this class of enzymes to try to get phenolic glucosides. First, the glucosyltransferase from Streptococcus sobrinus (referenced by the authors as strain 6715, serotype g) was proven to catalyze the synthesis of 4′-O-α-D-glucopyranosyl-(+)-catechin in a strictly aqueous medium (catechin at 1 g/L in 100 mM phosphate buffer pH 6.0 containing 2% sucrose) (NAKAHARA K, KONTANI M, ONO H, KOMADA T, TANAKA T, OOSHIMA T, HAMADA S (1995) Glucosyltransferase from Streptococcus sobrinus catalyzes glucosylation of catechin. Appl. Environ. Microbiol. 61(7): 2768-70). A similar enzyme, the glucosyltransferase-D from Streptococcus mutans GS-5, was proven to be less regioselective, as it catalyzes not only the synthesis of 4′-O-α-D-glucopyranosyl-(+)-catechin but also the synthesis of 7-O-α-D-glucopyranosyl-(+)-catechin and of the diglucosylated derivative 4′,7-O-α-D-diglucopyranosyl-(+)-catechin (MEULENBELD G H, ZUILHOF H, VAN VELDHUIZEN A, VAN DEN HEUVEL RHH, HARTMANS S (1999) Enhanced (+)-catechin transglucosylating activity of Streptococcus mutans GS-5 glucosyltransferase-D due to fructose removal. Appl Environ Microbiol 65(9): 4141-7). Though several investigations regarding the acceptor specificity of Streptococcus mutans GS-5 glucosyltransferase lead the authors to infer that aromatic acceptors appear to require two adjacent aromatic hydroxyl groups (MEULENBELD G H, HARTMANS S (2000) Transglycosylation by Streptococcus mutans GS-5 glucosyltransferase-D: acceptor specificity and engineering reaction conditions. Biotechnol Bioeng 70(4): 363-9), this statement was counteracted by the identification of glucosylation at position 7 in catechin (MEULENBELD et al., 1999) and by the synthesis of non-pyrocatechol derivatives. Indeed, pinosylvin and resveratrol, respectively 3,5-dihydroxy-trans-stilbene and 3,4′,5-tri hydroxy-trans-stilbene, were glucosylated by a crude glucosyltransferase preparation produced by Streptococcus mutans to form respectively 3-O-α-D-glucopyranosyl-(E)-pinosylvin and 3-O-α-D-glucopyranosyl-(E)-resveratrol (SHIM H, HONG W, AHN Y (2003) Enzymatic preparation of phenolic glucosides by Streptococcus mutans. Bull Korean Chem Soc 24(11): 1680-2). Very recently, it was claimed that the flavonols quercetin and myricetin and the flavone luteolin could be glucosylated by special glucansucrases, namely the Leuconostoc mesenteroides NRRL B-512F dextransucrase (sucrose: 1,6-α-D-glucan 6-α-D-glucosyltransferase, EC 2.4.1.5) and the Leuconostoc mesenteroides NRRL B-23192 alternansucrase (sucrose:1,6(1,3)-α-D-glucan 6(3)-α-D-glucosyltransferase, EC 2.4.1.140) (BERTRAND A, MOREL S, LEFOULON F, ROLLAND Y, MONSAN P, REMAUD-SIMEON M (2006) Leuconostoc mesenteroides glucansucrase synthesis of flavonoid glucosides by acceptor reactions in aqueous-organic solvents. Carbohydr Res 341: 855-63). Conventionally, in the presence of sucrose, the former produces a glucan (dextran) in which 95% of the glucosidic bonds are α-(1→6) (skeleton of the polysaccharide) and 5% α-(1→3) (branching points), and the latter a glucan (alternan) in which the glucosidic bonds are alternatively α-(1→6) and α-(1→3). The obtained flavonoid derivatives were: luteolin-3′-O-α-D-glucopyranoside, luteolin-4′-O-α-D-glucopyranoside, quercetin-3′-O-α-D-glucopyranoside, quercetin-4′-O-α-D-glucopyranoside, quercetin-3′-4′-O-α-D-diglucopyranoside, myricetin-3′-O-α-D-glucopyranoside and myricetin-4′-O-α-D-glucopyranoside. This work demonstrates that yields of glycoside derivative synthesis not only rely on the enzyme itself (the synthesis of luteolin-O-glycosides dropped down from 44% to 8% between dextransucrase and alternansucrase), but also on slight chemical differences between two acceptors (no conversion was observed with the dextransucrase on diosmetin and diosmin).
From the above significant (though not exhaustive) state of the art regarding the experimented ways to obtain glucosylated derivatives of polyphenols in general (and flavonoids in particular) in order to overcome the main conventional drawbacks of flavonoids (poor water solubility at physiological conditions, in particular at pH ranging from 5 to 7 and 30° C. and high sensitivity to autoxidation in these biological conditions), it clearly appears that no precise guidelines can be deduced to set up the enzymatic production of a specific phenolic glycoside. On the contrary, it shows that there is no way for a man of the art to predict which flavonoid can be glucosylated with which enzyme and in which conditions to obtain high glucoside concentrations (see summary in Table 2). Indeed, though attempts have been made to establish a relationship between the phenolic structures and the possibility of their use as glycosyl acceptors by glycosyltransferases, it still appears that the obtention of glycosylated phenolics strongly depends on the nature of the phenolic substance and on the enzyme used for the condensation reaction. This is particularly true with glucosyltransferases conventionally synthesizing α-D-glucans from sucrose (EC 2.4.1.5), for which only a very small number of polyphenolic structures have been successfully reported. Furthermore, in the case of the main glucosyltransferases studied, namely S. mutans GS-5 glucosyltransferase D and L. mesenteroides NRRL B-512F dextransucrase, it has to be mentioned that the former synthesizes a water-soluble α-glucan in a primer-stimulated and dependent manner (HAMADA N, KURAMITSU H K (1989) Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis. Infect Immun 56: 1999-2005) whereas the later does not (ROBYT J F, WALSETH T F (1978) the mechanism of acceptor reactions of Leuconostoc mesenteroides NRRL B-512F. Carbohydr Res 61: 433-45). These glucosyltransferases have distinct mechanisms of action and consequently molecules that are acceptors for an enzyme are not necessarily acceptors for another; in other words, as shown in the previously cited works, there is no justification to consider that the substances that act as glucosyl acceptors in the case of S. mutans GS-5 glucosyltransferase D act also as glucosyl acceptors in the case of L. mesenteroides NRRL B-512F dextransucrase and vice versa.
All the more, prior art information shows that despite the interest and abundance of phenolics, few phenolic glycosides have been obtained by enzymatic reactions.
TABLE 2POLYPHENOLENZYME ORIGINPRODUCT(S) AND REFERENCEEnzymes and substrates: Glycosyltransferases able to transfer the sugar moiety of a sugarnucleotide (e.g. UDP-glucose)Caffeic acidArabidopsis thalianaCaffeoyl-3-O-β-glucoside - LIM et(OH in 3 and 4)al. 2003o- and m-coumaric acidsArabidopsis thaliana2-O- and 3-O-β-glucosides of o-(OH in 2 and 3, respectively)and m-coumaric acids - LIM et al.2003Isoferulic acidArabidopsis thaliana3-O-β-glucoside - LIM et al. 2003(OH in 3; OCH3 in 4)p-coumaric acid (OH in 4), ferulicArabidopsis thalianaNo glucoside - LIM et al. 2003acid (OH in 4 and OCH3 in 3) andsinapic acid (OH in 4 and OCH3 in 3and 5)Quercetin (flavonol; OH in 3, 5, 7, 3′Arabidopsis thaliana3-O-, 7-O-, 3′-O-, 4′-O-and 4′)monoglucosides and 3,7-di-O and7-3′-di-O-glucosidesLIM et al. 2003; LIM et al. 2004Luteolin (flavone; OH in 5, 7, 3′ andArabidopsis thalianaGlucosides - LIM et al. 20034′)Eriodictyol (flavanone; OH in 5, 7, 3′Arabidopsis thalianaNo glucoside - LIM et al. 2003and 4′)Catechin (flavanol; OH in 3, 5, 7, 3′Arabidopsis thalianaNo glucoside - LIM et al. 2003and 4′) and cyanidin (anthocyan; OHin 5, 7, 3′, 4′)Enzymes and substrates: Starch degrading enzymes (α-glucosidase, cyclodextringlucanotransferase or CGTase, α-amylase) and starch and/or starch hydrolyzatesQuercetin (flavonol; OH in 5, 7, 3′α-glucosidase: pig liver,a-glucosyl quercetin (U.S. Pat. No. 5,565,435)and 4′)buckwheat seed, Mucor,(OH glucosylated not mentioned)Penicillium,SaccharomycesCGTase: Bacillus,Klebsiellaα-amylase: AspergillusEnzymes and substrates: Glycosyltransferases able to transfer the glucose moiety of sucroseCatechin (flavanol; OH in 3, 5, 7, 3′Streptococcus sobrinus4′-O-α-D-glucopyranosyl-(+)-and 4′)catechin (NAKAHARA et al. 1995)Resveratrol (OH in 3, 5, 4′) andStreptococcus mutans3-O-α-D-glucopyranosyl-(E)-pinosylvin (OH in 3, 5)pinosylvin and 3-O-α-D-glucopyranosyl-(E)-resveratrol(SHIM et al. 2003)Catechin (flavanol; OH in 3, 5, 7, 3′Streptococcus mutans4′-O-α-D-glucopyranosyl-(+)-and 4′)GS-5 (glucosyl-catechin, 7-O-α-D-glucopyranosyl-transferase D)(+)-catechin and 4′,7-O-α-D-diglucopyranosyl-(+)-catechin(MEULENBELD et al. 1999)Catechol (OH in 1 and 2), 3-Streptococcus mutansGlucosides (MEULENBELD andmethoxycatechol (OCH3 in 3), 3-GS-5 (glucosyl-HARTMANS, 2000)methylcatechol (CH3 in 3), 4-transferase D)methylcatechol (CH3 in 4)Phenol, 3-hydroxyphenol,Streptococcus mutansNo glucoside (MEULENBELD andbenzylalcohol, 2-hydroxybenzylGS-5 (glucosyl-HARTMANS, 2000)alcohol, 2-methoxybenzyl alcohol, 1-transferase D)phenyl-1,2-ethanediolQuercetin (flavonol; OH in 3, 5, 7, 3′L. mesenteroides NRRLGlucosides (3′ and 4′ with luteolinand 4′), luteolin (flavone; OH in 5, 7,B-512Fand L. mesenteroides NRRL B-3′ and 4′), myricetin (flavonol; OH inL. mesenteroides NRRL512F)3, 5, 7, 3′, 4′ and 5′)B-23192(BERTRAND et al. 2006)Diosmetin (flavone; OH in 5 and 3′,L. mesenteroides NRRLNo glucoside (BERTRAND et al.OCH3 in 4′)B-512F2006)L. mesenteroides NRRLB-23192
Another key point to consider in the enzymatic synthesis of phenolic glycosides is the possibility to create phenolic derivatives that enable recovering of the initial phenolics by a hydrolysis reaction in smooth conditions.
Indeed, for a given polyphenol, the advantageous properties that are presently known correspond to a specific structure and it has thus to be demonstrated that the valuable derivative with increased water solubility and stability properties can be converted into the saccharide part on one hand and the aglycone part on the other hand. One example of decrease of antioxidant activity due to glycolation is given by MISHRA et al. (MISHRA B, PRIYADARSINI K I, KUMAR M S, UNNIKRISHNAN M K, MOHAN H (2003) Effect of O-glycosylation on the antioxidant activity and free radical reactions of a plant flavonoid, chrysoeriol. Bioorg Med Chem 11: 2677-85). Chrysoeriol and its glycoside (chrysoeriol-6-OG-acetyl-4′-β-D-glucoside) are two flavonoids extracted from the tropical plant Coronopus didymus; chrysoeriol shows a better protective effect than the glycoside when tested for their ability to inhibit lipid peroxidation induced by gamma-radiation, Fe (III) and Fe (II). To date, this reversibility is only known for the α-glucosyl quercetin obtained with starch-degrading enzymes in vitro (U.S. Pat. No. 5,565,435). So, if the functionalization of phenolics as glycoside derivatives is a way (i) to facilitate their formulation in cosmetic, pharmaceutical or any other man-made preparations due to a higher water solubility than that of the aglycone and (ii) to increase the stability of these phenolics in said formulas, both of them being universal properties of the glucosylated forms of polyphenols, these glycoside derivatives must be hydrolyzable in biological conditions.
There is therefore a need to create:                new derivatives of valuable phenolic compounds with increased water solubility (in the same physico-chemical conditions (pH, salinity, temperature, etc.)) and stability; and/or        new derivatives of valuable phenolic compounds that can be readily converted into their precursor, glucose and phenolic substance, in the place where they have to exert their biological activity and not during their storage in a commercial formula; and/or        new derivatives of valuable phenolic compounds that can be obtained through a process in which the synthesis and purification steps can be carried out in a reproducible manner and at any scale dependent on the market demand.        
Owing to the fact that the pyrocatechol structure (presence of two vicinal hydroxyl groups) is recognized as particularly important for the scavenging activity of polyphenols, the phenolic compounds that seem particularly efficient are those containing a catechol structure; among the phenolic compounds that are of particular interest, there are the following compounds:                protocatechuic acid (3,4-dihydroxybenzoic acid, FIG. 2) and its ester derivatives; and/or        caffeic acid (3,4-dihydroxycinnamic acid, FIG. 3) and its ester derivatives, especially rosmarinic acid (3,4-dihydroxycinnamic acid (R)-1-carboxy-2-(3,4-dihydroxyphenyl) ethyl ester), chlorogenic acid (3-O-(3,4-dihydroxycinnamoyl)-D-quinic acid), chicoric acid, echinacoside, verbascoside and caffeic acid phenethyl ester, and its reduced form, hydrocaffeic acid, and its ester derivatives; and/or        special structures not closely related to protocatechuic acid or caffeic acid and containing the pyrocatechol ring: 3,4-dihydroxymandelic acid (FIG. 4) and its related substance 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylglycol with a C2-C6 skeleton, and esculetin (6,7-dihydroxycoumarin, FIG. 5) with a C6-C3 skeleton; and/or        the flavanones taxifolin (3,5,7,3′,4′-pentahydroxyflavanone, FIG. 6), fustin (3, 7,3′,4′-tetrahydroxyflavanone), eriodictyol (5,7,3′,4′-tetrahydroxyflavanone); and/or        the flavonols fisetine (3,7,3′,4′-tetrahydroxyflavone) and rhamnetin (3,5,3′,4′-tetrahydroxy-7-methoxyflavone); and/or        the flavones cirsiliol and 3′,4′,7-trihydroxyflavone and the isoflavone 3′-hydroxydaidzein.        
More detailed information on these phenolics of interest is included below.
Protocatechuic acid (also named 3,4-dihydroxybenzoic acid) is found in many edible and medicinal plants, though most of the time at concentrations lower than derivatives of cinnamic acid. Though slightly less potent than caffeic acid, protocatechuic acid showed a time-dependent and dose-dependent inhibitory effect on T47D human breast cancer cell growth. It was also demonstrated that protocatechuic acid and caffeic acid interact directly with the aryl hydrocarbon receptor, inhibit nitric oxide synthase and have a pro-apoptotic effect (KAMPA M, ALEXAKI VI, NOTAS G, NIFLI A P, NISTIKAKI A, HATZOGLOU A, BAKOGEORGOU E, KOUIMTZOGLOU E, BLEKAS G, BOSKOU D, GRAVANIS A, CASTANAS E (2004) Antiproliferative and apoptotic effects of selective phenolic acids on T47D human breast cancer cells: potential mechanisms of action. Breast Cancer Res 6: R63-R74). LIU et al. (LIU K S, TSAO S M, YIN M C (2005) In vitro antibacterial activity of roselle calyx and protocatechuic acid. Phytother Res 19(11): 942-5) demonstrated in vitro an inhibitory effect of protocatechuic acid on the growth of methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. The data from inhibition zone and minimum inhibitory concentration (MIC) values showed that protocatechuic acid effectively inhibited the growth of all tested bacterial pathogens. Recent studies indicate that protocatechuic acid could be used as a protective agent against cardiovascular diseases and neoplasms (SZUMILO J. (2005), Postepy Hig Med Dosw (Online) 59: 608-15). The mechanism of its action is mostly associated with antioxidant activity, including inhibition of generation as well as scavenging of free radicals and up-regulating enzymes which participate in their neutralization.
It was also demonstrated that protocatechuic acid is a possible chemopreventive agent for colon carcinogenesis through the suppression of manifestation of intermediate biomarkers induced by azoxymethane (AOM)-induced colon carcinogenesis in rats (TANAKA T, KOJIMA T, SUZUI M, MORI H. (1993) Chemoprevention of colon carcinogenesis by the natural product of a simple phenolic compound, protocatechuic acid: suppressing effects on tumor development and biomarker expression of colon tumorigenesis. Cancer Res. September 1; 53(17): 3908-13). Protocatechuic acid is therefore also a valuable active phenolic compound, but its bioavailability should be increased through functionalization to obtain more water-soluble derivatives.
Caffeic acid (also named 3,4-dihydroxycinnamic acid), a derivative of trans-cinnamic acid (trans-3-phenylacrylic acid), contains a —CH═CH—COOH group which ensures greater H-donating ability and subsequent radical stabilization than the carboxylate group in benzoic acids (RICE-EVANS C A, MILLER N J, PAGANDA G (1996) Structure—antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 20(7): 933-56). In addition to its possible beneficial effects on human health (caffeic and 3-methoxycaffeic or ferulic acids react with nitrite in vitro and inhibit nitrosamine formation in vivo; they also inhibit tyrosine nitration mediated by peroxynitrite), caffeic acid recently proved effective in protecting human skin from UVB-induced erythema (SVOBODOVA A, PSOTOVA J, WALTEROVA D (2003) Natural phenolics in the prevention of UV-induced skin damage. A review. Biomed Papers 147: 137-145). Caffeic acid is frequently encountered in the form of derivatives, with 1-carboxy-2-(3,4-dihydroxyphenyl)-ethanol to form rosmarinic acid, quinic acid to form chlorogenic acid and phenylethanol to form caffeic acid phenethyl ester.
Rosmarinic acid (also named 3-(3,4-dihydroxyphenyl)-2-[3-(3,4-dihydroxyphenyl)prop-2-enoyloxy]propanoic acid) is found in the Lamiaceae genus of plants, which includes basil, sage, mint, rosemary and perilla leaf (AL SEREITI M R, ABU-KAMER K M, SEN P (1999) Pharmacology of rosemary and its therapeutic potentials. Indian J. Exp Biol 37(2): 124-30). Oral supplementation with perilla leaves or extracts of rosmarinic acid has been shown to suppress allergic reactions in mice and, more recently, in humans (MAKINO T, FURUTA A, FUJII H, NAKAGAWA T, WAKUSHIMA H, SAITO K, KANO Y (2001) Biol Pharm Bull 24(10): 1206-9—TAKAKANO H, OSAKABE N, SANBONGI C, YANAGASIWA R, INOUE K I, YASUDA A, NATSUME M, BABA S, ICHIISHI E I, YOSHIKAWA T (2004) Extract of Perilla frutescens enriched for rosmarinic acid inhibits seasonal allergic rhinoconjunctivitis in humans. Exp Biol Med 229(3): 247-54). Rosmarinic acid relieves allergy symptoms by preventing the activation of immune responder cells and by inducing apoptosis, or cellular suicide, in already activated immune responder cells (HUR Y G, YUN Y, WON J (2004) Rosmarinic acid induces p561ck-dependent apoptosis in jurkat and peripheral T cells via mitochondrial pathway independent from fas/fas ligand interaction. J Immunol 172(1): 79-87). Rosmarinic acid has also been shown to kill allergy-activated T cells and neutrophils during allergic reactions without affecting the T cells or neutrophils in their resting state (SANBONGI C, TAKANO H, OSAKABE N (2003) Rosmarinic acid inhibits lung injury induced by diesel exhaust particles. Free Radic Biol Med 34(8): 1060-9).
Rosmarinic acid was first shown to reduce allergic reactions in mice using the mouse ear passive cutaneous anaphylaxis reaction (MAKINO T, FURATA Y, WAKUSHIMA H, FUJII H, SAITO K, KANO Y (2003) Anti-allergic effect of Perilla frutescens and its active constituents. Phytother Res 17(3): 240-3). One study showed that rosmarinic acid inhibited IL-2 promoter activation of T cells in a large-scale screening of plant extracts (WON J, HUR Y G, HUR E M, PARK S H, KANG M A, CHOI Y, PARK C, LEE K H, YUN Y (2003), Rosmarinic acid inhibits TCR-induced T cell activation and proliferation in a Lck-dependent manner. Eur J Immunol 33(4): 870-9). Another study showed that rosmarinic acid, by inhibiting both the activation and proliferation of T cells, had potent immunosuppressive effects when combined with rapamycin, an anti-rejection drug (YUN S Y, HUR Y G, KANG M A, LEE J, AHN C, WON J (2003) Synergistic immunosuppressive effects of rosmarinic acid and rapamycin in vitro and in vivo. Transplantation 75(10): 1758-60).
Chlorogenic acid (also named 1,3,4,5-Tetrahydroxycyclohexanecarboxylic acid 3-(3,4-dihydroxycinnamate)) is the major soluble phenolic in solanaceous species such as potato, tomato and eggplant. It also accumulates to substantial levels in apples, pears, plums and coffee. SAWA et al. (SAWA T, NAKAO M, AKAIKE T, ONO K, MAEDA H (1999) Alkylperoxyl radical-scavenging activity of various flavonoids and other phenolic compounds: implications for the anti-tumor prompted effect of vegetables. J Agric Food Chem 47: 397-402) observed that it removes particularly toxic reactive species by scavenging alkylperoxyl radicals and may prevent carcinogenesis by reducing the DNA damage they cause.
Caffeic phenethyl ester (CAPE) is one of the major components of honeybee propolis, the resinous dark-colored material which is collected by honeybees from the buds of living plants mixed with beeswax and salivary secretions. CAPE is a potent and a specific inhibitor of activation of members of the transcription factor NF-κB family and this may provide the molecular basis for its multiple immunomodulatory and anti-inflammatory activities (NATARAJAN K, SINGH S, BURKE T R, GRUNBERGER D, AGGARWAL B B (1996) Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-κB. Proc Natl Acad Sci USA 93: 9090-5). More recently, the role of CAPE as a potent antimetastatic agent which can markedly inhibit the metastatic and invasive capacity of malignant cells was evidenced (HWANG H J, PARK H J, CHUNG H J, MIN H Y, PARK E J, HONG J Y, LEE S K (2006) Inhibitory effects of caffeic acid phenethyl ester on cancer cell metastasis mediated by the down-regulation of matrix metalloproteinase expression in human HT1080 fibrosarcoma cells. J Nutri Biochem 17: 356-62).
Esculetin (or aesculetin, also named 6,7-dihydroxycoumarin), a member of the family of the C6-C3 phenolics, has a coumarin structure derived from trans-cinnamic acid via ortho-hydroxylation (for memory, caffeic acid is 3,4-dihydroxycinnamic acid), trans-cis isomerisation of the side chain double bond and lactonisation. Whereas the trans form is stable and cannot cyclize, the cis form is very unstable and cyclization is thus favored. Glucose is a good leaving group which assists in the cis-trans transformation. A specific enzyme found in Melilotus alba (Leguminosae) specifically hydrolyses the cis-glucoside glucosidase). Some of its properties are the inhibition of Ras-mediated cell proliferation and attenuation of vascular restenosis following angioplasty in rats (PAN S L, HUANG Y W, GUH J H, CHANG Y L, PENG C Y, TENG C M (2003) Esculetin inhibits Ras-mediated cell proliferation and attenuates vascular restenosis following angioplasty in rats. Biochem Pharmacol 65: 1897-1905) and the inhibition of mushroom tyrosinase (MASAMOTO Y, ANDO H, MURATA Y, SHOMOISHI Y, TADA M, TAKAHATA K (2003) Mushroom tyrosinase inhibitory activity of esculetin isolated from seeds of Euphorbia lathyris L. Biosci Biotechnol Biochem 67(3): 631-4). It has to be mentioned that esculetin is frequently encountered as a glucoside, esculin (esculetin-6-β-D-glucopyranoside), with a β-glucosidic linkage at position 6. The members of the C6-C2 phenolics are basically found in the catecholamine metabolism and 3,4-dihydrophenyl related substances could have interesting properties (EISENHOFER G, KOPIN I J, GOLDSTEIN D S (2004) Catecholamine metabolism: a contemporary view with implications for physiology and medicine. Parmacol Rev 56(3): 331-49).
Taxifolin (or dihydroquercetin, or 3,5,7,3′,4′-pentahydroxyflavanone, or (2R,3S)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-chroman-4-one) occurs in various barks (Larix sibirica Lebed, Pinus pinaster ssp. atlantica) and in Silybum marinum seeds (used for the preparation of the silymarin complex and containing silymarin flavonolignans which are biogenetically formed by oxidative addition of coniferyl alcohol to taxifolin. It has a chiral bond between cycle B and the two other cycles. Relating to the PP-vitamin group, it possesses a wide spectrum of biological activities (MIDDLETON E, KANDASWAMI C, THEOHARIDES T C (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacol Rev 52(4): 673-752). It shows capillary-protecting, anti-inflammatory and gastro-protective action, decreases spasms of sleek muscles of the intestine, increases function of the liver and possesses antiradiation protective activity. Taxifolin has also been shown to have potential applications in reducing skin inflammation (BITO T, ROY S, SEN CK, SHIRAKAWA T, GOTOH A, UEDA M, ICHIHASHI M, PACKER L (2002) Flavonoids differentially regulate IFN-gamma-induced ICAM-1 expression in human keratinocytes: molecular mechanisms of action. FEBS Lett. 520(1-3): 145-52). However, Taxifolin is poorly soluble in aqueous solution (around 1 g/l), which prevents its usage for some cosmetic and therapeutic applications.
Glycosylation being recognized to render polyphenols, in vegetal cells as well as in vitro, more water-soluble and less reactive toward free radicals, if glucosides of these phenolics of particular interest exist, then they might represent polyphenol derivatives with increased water solubility and stability, and thus with increased added value.
It would also be useful to obtain derivatives from these phenolics which can be converted during their final usage in the metabolizable initial phenolic structure. This objective can be achieved by means of the present invention.